1. Field of the Invention
The invention relates to a device for selecting stem cells with a serum free medium for proliferation of stem cells. The present invention also relates to a method of treating or preventing diseases caused by cancer-related stem cells comprising administrating a therapeutically effective amount of resveratrol.
2. Description of the Related Art
Progenitor cells, also called stem cells, indicate the cells in original and retain the ability to renew themselves through mitotic cell division and can differentiate into a diverse range of specialized cell types. Research in the stem cell field grew out of findings by Canadian scientists Ernest A. McCulloch and James E. Till in the 1960s. According to the potency of differentiation, all the stem cells can basically be divided into three kinds of types: the first kind of type is the totipotent stem cells, which by themself are able to give rise to an entire embryo and have the very strong differentiation ability to prolify infinitly and form multiple cells, tissues, organs, and even individuals. The second kind of type is the pluripotent stem cells, this kind of stem cell also has the potency to specialize into many kinds of cells or tissues. The difference between the second kind and the preceding one is that the pluripotent stem cells are not capable of develop a whole individual and the potential growth is limit in some way. The third kind of type is the unipotent stem cells, which are only able to progress differentiation of one function or two functions cells with closed-relationship. Traditionally, people assume that neural stem cells and so on can only transform into neurons with closed functions like astrocytes and oligodendrocytes. However, the transdifferentiation studies demonstrate that neural stem cells also have the ability of differentiation being hematopoietic cells. Furthermore, rely on the source of the stem cells, all of them can be group into inner cell mass or embryonic germ cells and undifferentiated adult stem cells gathered from tissues. The former comes from teratoma, morula, inner cell mass, embryoid bodies or progenitor cells. The latter comes from neuron, blood, meschymal, epidermal or lipid.
Recently, more and more studies demonstrate that the cells isolated from partial tumor tissues also possess the characteristics of stem cells. Back to 1937, Jacob Furth and colleagues showed that leukemia can be transmitted from one mouse to another using a single undifferentiated leukemia cell. In 1963, Robert Bruce and Hugo Van der Gaag used the spleen colony-forming assay (CFU-S)—a tool first developed by James Till and Ernest McCulloch, and now widely used in stem-cell biology—to show that only a small subset of primary cancer tissue was able to proliferate in vivo. Collectively, these studies underscored the functional heterogeneity in tumours—not every cell is able to proliferate to form a colony in vitro or to give rise to a tumour when transplanted in vivo—and introduced the concept of CSCs. Thus, the CSCs have been proved from the tissues of leukemia, brain tumor, nasopharyngeal carcinoma, lung tumor and so on. (Bergsagel D. E. et al., Cancer Res, 1968, vol. 28, 2187-96 ∘ Park C. H. et al., J Natl Cancer Ins, 1971, vol. 46, 411-22 ∘ Heppner G. H. et al., Cancer Res, 1984, vol. 44, 2259-65).
Cancer-related stem cells are a sub-population of cancer cells that possess characteristics normally associated with stem cells. These cells are believed to be tumorigenic (tumor-forming), in contrast to the bulk of cancer cells, which are thought to be non-tumorigenic. Cancer-related stem cells have stem cell properties such as self-renewal and the ability to differentiate into multiple cell types. A theory suggests such cells persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors. Development of specific therapies targeted at cancer-related stem cells holds hope for sufferers of metastatic disease.
While stem cells are best defined functionally, a number of molecular markers have been used to characterize various stem cell populations. Although functions have yet to be ascertained for many of these early markers, their unique expression pattern and timing provide a useful tool for scientists to initially identify as well as isolate stem cells.
Cancer-related stem cells are extracted from the organs of adult individuals. At present believed that, the molecular markers and cellular mophology of the cancer-related stem cells are similar with those of the adult stem cells. Detection of surface antigens or specific transcriptional factors is used as molecular markers. Generally acknowledged markers are briefly being classified as four groups as followed. The first subset used to detect the embryonic stem cells includes surface markers such as stage specific embryonic antigen 3 (SSEA3), stage specific embryonic antigen 4 (SSEA4), tumor resistant antigen-1-60 (Tra-1-60) and tumor resistant antigen-1-81 (Tra-1-81) and stemness genes such as IPS-Oct4, Nanog, SOX2, Klf-4, c-Myc and Lin28. The second subset used to affirm the existence of hematopoietic stem cells includes CD34, CD133 and ATP-binding cassette superfamily G member 2 (ABCG2). The third subset used to confirm whether neural stem cells comprises nestin, polysialic acid-neural cell adhesion molecule (PSA-NCAM) and p75 neurotropin R (NTR). The forth subset required to recognize mesenchymal stem cells is STRO-1, an antibody to identify stromal precursors.
Muhammad figured out the specific surface molecular markers aimed at breast cancer-related stem cells, such as CD44, CD24, B38.1 and epithelial specific antigen (Muhammad A. H. et al., 2003, Proc. Natl. Acd. Sci. USA, vol. 100, 3989-88). Other scientists also indicated CD133 as the marker of colon or prostate cancer-related stem cells (Catherine A. O. et al., Nature, 2007, vol. 445, 106-110 ∘ Gavin D. et al., J Cell Sci, 2003, vol. 117, 3539-45). Besides, Wolf suggested that side population cells is the characteristic of cancer-related stem cells (Wolf N. S. et al., Exp Hematol, 1993, vol. 21, 614-22 ∘ Wolf N. S. et al., Blood, 2001, vol. 98, 1166-73). Moreover, Max S. Wicha and his colleagues utilized aldehyde dehydrogenase 1 (ALDH-1) as a marker to identify normal or malignent human cancer-related stem cells. However, as to stemness genes detection, the genes mentioned above could be proved in the following references, such as Chamber I. et al., Cell, 2003, vol. 113, 643-55; Mitsui K. et al., Cell, 2003, vol. 113, 631-42; Schöler, H. R. et al., Trends Genet, 1991, vol. 7, 323-329; Maurizio P et al., Stem Cells, 2001, vol. 19, 271-8).
Isolation and cultivate stem cells after identifying the characteristic of them for sequential basic research of gene regulation, human repair or the application of being drug candidate screening platform. Selective culture medium provides cells not only an amount of nutrients to replicate, but also acts as a screening tool of identifying subsets toward particular differentiation pathway. Take neural stem cells for example, cells was cultured with serum free medium containing insulin, transferrin, sodium selenite, fibroblast growth factor, fibronectin and so on for increasing a great amount of neural epithelial progenitor cells possessing the expression of nestin. Besides, sonic hedgehog was added to the cultured differentiated neural cells and led cells tend to augment numbers of cells expressing NKx6.1 and Olig2, the characteristic of ventral lateral neurons.
In some embodiments, embryonic stem cells were cultivated as embryoid bodies for four days with the medium further containing fibronectin. Therefore, approximately 85% of all like neural epithelial cells subset in morphology expressed the surface marker of neural epithelial cells, nestin. Furthermore, to add fibroblast growth factor 2 would promote cellular survival and amplification greatly, but enhance the neural cells to differentiate. If removal of fibroblast growth factor 2, there would be a great amount of cells dead. Treat cell as above, another problem presented was that there was still expression of epithelial cells' marker, such as cytokeratin 18. At the same time, pluripotent stem cells present and could be recognized from some cellular population by detecting whether stage specific embryonic antigen 1 or not.
Another method used medium to selectively promote neural cells tend to differentiate was treating embryonic stem cells in nearly low cells density with leukemia inhibitory factor. Like this, we could get neural cell clusters comprising about 100% neural progenitor cells expressing nestin. Some researchers were under the impression that such cells were neural stem cells because this kind of cells would response to leukemia inhibitor factor, but fibroblast growth factor 2. Another interesting opinion was that retinol acid is not apparently essential to differentiated neural cells. Back to the words, treating cells only with leukemia inhibitory factor would get low survival rate of its, about one of 2000. Hence, such method is not a normal approach to differentiate neural cells.
Cytokines is considered to be related closely to amplification and differentiation of neural stem cells. Several cytokines might induce the important activated differentiation. Nevertheless, there is no such cytokine able to induce neural progenitor cells to transform into functional stem cells in vitro. Interleukins are some of them, such as interleukin 1, interleukin 7, interleukin 9, interleukin 11 and so on. Neurotrophie factor can affect whole processing of differentiation. If treat neural stem cells with brain-derived neurotrophie factor, plenty of them will possess the characteristic of neurons. As to growth factors, such as epithelial growth factor, neural growth factor, or basic fibroblast growth factor, they also influence the differentiation of neural stem cells. The responses to assorted, various concentrations and multiple combined treating are distinct from each others. Even the same factors applied to different stages of development and differentiation of neural stem cells, they cause diverse regulations. Therefore, to cultivate cells in the presence of epithelial growth factor and basic fibroblast growth factor would direct embryonic neural stem cells to develop toward neurons, astrocytes and oligodendrocytes. Fetal and adult brain neural stem cells, treated with or without epithelial growth factor and basic fibroblast growth factor developed majorly into astrocytes. These studies suggested that the epithelial growth factor or basic fibroblast growth factor-induced differentiation is complicated. Besides, referring to chemicals, retinoic acid is commonly used on account of its important roles in the process of embryo growth, especially in the development of neurons.
Related techniques derived from selectively culturing cancer-related stem cells are compared as following.
Human breast tumor tissues after cellular matrix being trypsinized were injected subcutaneously into severe combined immunodeficient mice as previous study described in Muhammad A. H. et al, PNAS, 2003, vol. 100, 3983-3988. Human cancer-related stem cells were able to form tumor tissues in mice in vivo. Afterwards, human cancer-related stem cells possessing the properties of highly differentiation and self-renewal were isolated from mice for only discussing the mechanism, but the procedure could not be done in a great quantity.
Cancer-related stem cells were selected from brain tumor primary tissue cultures with cancer spheroid cells selecting medium and the methodology was published by Sheila K. S. and his associates in the Chinese journal of Cancer Research in the same year. In brief, the scientific group provided us a steady and effective selective culture medium in vitro, a serum-free medium containing 20 ng/ml human recombinant epithelial growth factor, 20 ng/ml basic fibroblast growth factor, 10 ng/ml leukemia inhibitory factor, 1× neural survival factor and 60 μg/ml acetyl cysteine. The disadvantage of its was so expensive that plenty of experiments used above recombinant proteins needed enough budgets (Sheila K. S. et al., Cancer Research, 2003, vol. 63, 5821-5828).
Another proof of stimulating the proliferation of endometrial stroma cells was cultivating isolated cells with 10 ng/ml lactoferrin in vitro (Atsushi Yanaihara et al., Molecular Human Reproduction, 2000, vol. 6, 469-473). Andrew Grey and his colleague promoted the differentiation and survival of osteoblasts by treating cells with lactoferrin in vitro (Andrew Grey et al., Molecular Endocrinology, 2004, vol. 16, 2268-2278). The medium containing 2% fetal bovine serum was supplied in previous reference, and medium containing 5% fetal calf serum was in latter one. The above two culturing systems were not suitable for highly throughput screening with such serum-contained medium.
Li, Ming-Chu and his fellow workers illustrated how to select and identify the cancer-related stem cells from primary human medulloblasts. In brief, cancer-related stem cells isolated from patients' tissues to be single-cell suspension followed by inoculating into the serum-free medium consisting of epithelial growth factor, fibroblast growth factor and B27 supplement to cultivate in suspension. In order to assertain the percentage of cancer-related stem cells, they tested mono-colony forming assay accompanied with subculturing spheroid cells continuously. Thereafter, the cancer-related stem cells grew in the medium with serum and the phenomenon were investigated (Li, M. C. et al., Cancer (in Chinese), vol. 2).
Ouyang, Zhen and his colleague obtained spheroid cells and subsequently cultured them with serum-free DMEM/F12K medium comprising 20 μg/ml epithelial growth factor, 20 μg/ml basic fibroblast growth factor, 2 μmole/L L-Glutamine, 4 U/L insulin, 100 U/mL Penicillin G and 100 U/mL Streptomycin with pH of 7.2 to 7.5. The Stro-1+ cells were isolated by immunomagnetic beads as cancer-related stem cells (Ouyang. Z. et al., Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu, 2007, vol. 11(24), 4706-4709).
Akio S. discussed how important the epithelial growth factor was in the progress of cells' proliferation. The culturing condition was serum-free DMEM/F12K medium containing streptomycin, penicillin G, B27 supplement, 20 μg/ml epithelial growth factor, 20 μg/ml fibroblast growth factor, 1000 U/ml leukemia inhibitory factor. Besides, the authors suggested that only epithelial growth factor could induce the formation of spheroid cells and increase the capability of self-renew (Akio S. et al., J. BioChem, 2008, vol. 3, 1-10).
Others desired to find out the ways of how to identify neural stem cells so as to have a cure for neuropathy or have an idea to discover the presence of brain tumors. Mouse neural stem cells were selected and grew in the NSC medium consisting of 20 μg/ml epithelial growth factor, 20 μg/ml fibroblast growth factor and 2 μg/ml heparin. Medium was exchanged at two or three day-interval (Phedias et al., Nature Chemical Biology, 2007, vol. 3, 268-273).
In conclusion, at present selective culture to screen cancer-related stem cells is nothing more than using medium consisting of salts, vitamins, amino acids and even further to add some cytokines to promote the amplification of cells. Thereafter, isolated by traditional Transwell® approach and then scratch them or by flow cytometry employed with immunomafnetic beads. As rapidly as the development of biotechnology, such as genetic engineering, embryonic engineering, cellular engineering, tissue engineering and so on, more and more in vitro methods present to satisfy predicted purpose. The major applications of stem cells were being supply sources for translating every kind of cells, tissues and organs, hence cell culturing methodology and device of being used conveniently, clear mechanism, speed and suitable for curing diseases or high throughout screening of new dug candidates will be the critical points of development and modification. However, recently thermoresponsive materials such as poly(N-isopropyl acrylamide) (PNIPAAm), was applied to tissue engineering. Harimoto M and his fellow workers disclosed a novel method of co-culturing double cellular layers in the reference named “Ectopic transplantation of hepatocyte sheets fabricated with temperature-responsive culture dishes” (Harimoto M et al., J Biomed Mater Res., 2002, 62(3):464-70). Another research group also claimed a method to rebuild the corneal epithelial cells by making use of the cellular film of bioengineering (Hsiue G H et al., Transplantation, 2006, 81(3):473-6). Besides, Japanese CellSeed company further applied the thermoresponsive materials into culturing devices, such as culturing dishes or microplates.
Drug candidates screening is one step of modern medicine research and development procedure to detect and acquire compounds possessing particular bioactivity. In more details, it is a process to select compounds targeting specific bio-function and owning high efficacy through experimentation normalized by standard operation protocols.
Screening model being used in the drug screening experiments as a model of pharmacology experiments. For acquiring standardization and quantify, animal studies were commonly used in traditional pharmacology experiments, but not drugs screening. In light of the difference of experimental models, drugs screening could be classified into biochemistry-level and cell-level screening. Cell-level screening model, a mode more approaching physical condition, applied cell culturing techniques to obtain the target cells, which were purposed to have some specific therapeutic reactions. Thereafter, those cells treated with the drug candidates to determine the bio-activity of them through assays similar with biochemistry-level detection. There were four factors which were not only the critical elements in the process of virtual drugs screening, but a bottleneck to limit the accuracy of it, to build a proper pharmacophore model, to test exactly, to predict the molecular structures of the target proteins, and to calculate the charge of the free energy between the candidates and target cells. Although the accuracy needed to be raised, the high speed and low-priced of it make itself to become one of the fast development drugs screening systems. Scientists further pointed out cancer-related stem cells could resist currently medical therapy and proceed DNA repair after radiotherapy. Deserved to be mentioned, above features could be observed in parent cells stronger and more frequently than daughter cells. A topic report in Nature Biotechnology addressed that pharmaceutical companies had been traced cancer-related stem cells. Studies on cancer-related stem cells had been one of the strategies of researching and developing the anti-cancer drugs in some biotechnology or pharmaceutical firms, such as GlaxoSmithKline, Geron corp. (LA, US), Stemline Therapeutics, Inc. (NY, US), OncoMed Pharmaceuticals, Raven Biotechnologies Inc., Arius Research Inc., and Immunocellular Therapeutics Ltd. GlaxoSmithKline' Tyverb, a breast cancer small molecule drug, got FDA's approval and is going to reach the market first (Charlei S., Nature Biotechnology, 2008, vol. 26, 366-367).
EP 0513896 B1 disclosed a novel medium for the preservation of live organs, biological tissues or cells. This medium is composed of a liquid biological nutrient base such as a cell culture medium, enriched by a small amount of peroxidase enzyme proteins such as lactoperoxidase and/or enzymatic proteins with ferriheme such as lactoferrin. In particular, the invention can be applied to the preservation of corneas, but there were not mentioned that the medium can be used to proliferating stem cells.
The purpose of this invention is to supply the cell culturing methodology of being used conveniently, clear mechanism, speed and suitable for curing diseases or high throughout screening of new dug candidates. All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
Tumor formation in vitro displayed significant resistance to radiotherapy. The expression of embryonic stem cell genes such as Oct-4 and Nanog have been correlated with tumorigenesis and self-renewing activity, and can affect some aspects of tumor behavior such as recurrence and resistance to therapy. Recently, the expression of Oct-4, Nanog, and was shown in cancer-related stem cells derived from human oral, breast, and brain tumors, suggesting that their expression may be implicated in self-renewal and tumorigenesis via activating downstream target genes (Zhang H et al., J cell Biocham, 2008, vol. 103, 709-718).
Resveratrol (3,4′,5-tri-hydroxy-trans-stilbene), a natural polyphenol, is mostly found in grapes, red wine, and peanuts (Bradamante S et al., Drug Rev, 2004, vol. 22, 169-188). It possesses several pharmacological effects that are closely related to health therapies including cardiac protection as well as anti-viral, anti-inflammatory, and anti-aging activities and lifespan extension. Importantly, recent researches demonstrated that resveratrol (RV) has an anti-cancer effect and inhibits tumorigenesis by inducing apoptosis via Fas-, P53-, and P21WAF/CIP1-mediated pathways (Atten M J et al., Invest New Drug, 2005, vol. 23, 111-119; Kuo P L et al., Life Sci, 2002, vol. 72, 23-24; Roccaro A M et al., Clin Cancer Res, 2008, vol. 14, 1849-1858). Furthermore, some reports indicated that RV can also increase radiosensitivity in several cancer cell lines including melanoma, cervix carcinoma, chronic myeloid leukemia (K-562), and multiple myeloma (IM-9) (Johnson G E et al., Apoptosis, 2008, vol. 13, 790-802; Baatout S et al., Int J Mol Med. 2004, vol. 13, 895-902).
U.S. Pat. No. 7,455,860 discloses “Dietary supplement formulation for controlling inflammation and cancer”, relating to a dietary supplement which is a phytochemical composition. This composition is capable of controlling inflammatory conditions and preventing and curing cancer in mammals. The composition comprises a synergistic mixture of standardized Boswellia extract, salts of glucosamine, and curcuminoids optionally containing bromelain, chondroitin, methylsulphonylmethane, resveratrol, extracts of white Willow and ginger, and quercetin.
U.S. Pat. No. 6,008,260 discloses “Cancer chemopreventative composition and method”, which relates to a composition and method of cancer chemoprevention. The composition and method utilize resveratrol as a cancer chemopreventative agent in mammals, including humans.
Previous studies suggested that resveratrol (RV) could also increase radiosensitivity via several mechanisms, including inactivation of NF-κB and increased S phase cell cycle arrest. Recent studies showed that RV-induced apoptosis not only inhibits tumor growth but also acts as a radiochemosensitizer for anti-cancer therapy (Johnson G E et al., Apoptosis, 2008, vol. 13, 790-802; Baatout S et al., Int J Mol Med, 2004, vol. 13, 895-902). However, the treatment of role of RV in cancer-related stem cell and RV-mediated radiosensitizing effects in the treatment of cancer-related stem cells were still undetermined.
Adult stem cells from bone marrow and other tissues have been shown to be able to differentiate into many types of cells, including osteocytes, chondrocytes, smooth muscle cells, hepatocytes, cardiomyocytes, neurons, and retinal cells. They are considered promising resources for restorative cell therapy of various diseases. Recently, Yamanaka and colleagues demonstrated that induced pluripotent stem (iPS) cells could be generated from mouse embryonic fibroblasts as well as from adult human fibroblasts via the retrovirus-mediated transfection of four transcription factors, i.e., Oct3/4, Sox2, c-Myc, and Klf4. These iPS cells are indistinguishable from embryonic stem (ES) cells in morphology, proliferative abilities, surface antigens, gene expressions, epigenetic status of pluripotent cell-specific genes, and telomerase activity. The major advantage of iPS cells over ES cells is that iPS cells can be derived from patient's own somatic cells, thereby avoiding immune rejection after transplantation and ethical concerns raised in ES cells. However, previous studies have shown that transplanted iPS cells are likely to from teratoma in vivo because of the genetic changes intrinsic to the iPS cells generation process may pose risk of enhancing tumorigenesis through both the introduced genes themselves and in theory via the potential changes at specific integration sites, a feature also found in ES cells. The ability to form teratomas in vivo has been a landmark and routine assay for evaluating the pluripotency of ES as well as iPS cells, however, teratoma formation from pluripotent stem cells is considered as an unacceptable obstacle for the application of stem cell therapy in regenerative medicine. Thus, the regenerative medicine field is faced with a dilemma situation in that if one seeks to make stem cells safer by lowering Myc levels, a tandem reduction in the “stemness” of those cells may prove inevitable. The same appears to be true for other master stem cell regulators such as KLF4. Lowered levels of KLF family members including KLF4 substantially impaired ESC pluripotency and self-renewal, forcing ESC to differentiate. In other words, it is very hard to preserve self-renewal and pluripotency while eliminating tumorigenicity. Therefore, measures to overcome the tumorigenicity of iPS cells are crucial for successful treatment of patients with iPS cells.